Oomycete pathogens encode RNA silencing suppressors

Oomycetes is a large group of fungal-like eukaryotic microorganism that includes some plants and animal pathogens. Plant diseases caused by oomycetes lead to significant economic losses every year.

RNA silencing has been show to play a role in plant defense mechanism against pathogen. Effectors of plant pathogen are a class of proteins that encoded by pathogen to facilitate host infection. One of the effector functions is to counter the plant defense mechanism by impairing the host RNA silencing. The functions of many of these eukaryotic effectors are still unknown.

Like other pathogens, Oomycetes secrete a variety of effector proteins into plant intracellular and intercellular spaces. In this paper, Qiao et al. found that Phytophthora effectors can act as a RNA silencing suppressor and increased plant susceptibility to disease.

The authors used a screening assay to check 59 RXLR effectors and found two effectors. They coexpressed individual effector with green fluorescent protein (GFP) by Agrobacterium infiltration in the leaves of tobacco. Low or no green fluorescence in infiltrated zone means GFP genes are silenced, while strong fluorescence indicates the effector is capable to suppress siRNA-mediated silencing. These two proteins were then named as Phytophthora suppressors of RNA silencing 1 and 2 (PSR1 and PSR2)

PSR1 was shown to strongly reduce the abundance of siRNA and suppressed the systemic silencing. PSR2 reduced lower level of siRNA and didn’t cause systemic silencing. However, the in vitro assay showed that PSR1 and PSR2 do not bind single-stranded or double-stranded 21-nt small RNAs. The authors hypothesized that these protein are likely to block small RNA biogenesis of host.

Through examination of Arabidopsis expressing PSR1, they found representative miRNAs and endogenous siRNAs showed an overall reduction. They found that pri-miRNAs were not affected, but pre-miRNAs have a mild reduction. This suggested that PSR1 inhibits DCL1-mediated processing of pri-miRNA because DCL1 play roles in processing pre-miRNA to pri-miRNA. DCL2 and DCL4 is required for siRNA biogenesis, therefore PSR1 may target multiple DCLs or common DCL cofactor(s). The PSR1 protein contained a nuclear localization signal (NLS) and localized in the nuclei. They found that this NLS is important for PSR1 activity.

The PSR2 expressing plants didn’t showed any abnormal phenotype and representative miRNA level were normal. The only difference in these plants was the lower level of two 21 nt trans-acting siRNA (ta-siRNAs). Their further investigation found that PSR2 didn’t interfere with AGO1 and suggest that it is likely to target the accumulation of specific ta-siRNA species at a downstream step(s).

Next, the authors transformed PSR1 and PSR2 into potato virus X (PVX). From the plant infection assay, they showed that the PVX-PSR1 and PVX-PSR2 are both relatively more virulence than the wild type PVX.  When they infected tobacco leaves expressing PSR1 and PSR2 with P. infestans, the leaves also more susceptible compare to plants receiving empty vector. This suggests that PSRs are important virulence factors.

In summary, this study proved that oomycetes pathogens used effector to facilitate infection by suppressing the host RNA silencing.

 

An Early-Branching Microbialite Cyanobacterium Forms Intracellular

Cyanobacteria are aquatic bacteria that are able to perform photosynthesis. It is suggested that cyanobacteria have existed on earth for at least 2000 million years. Cyanobacteria play important role in the global carbon cycle. They transform carbon dioxide into organic carbon and carbonates.

The cyanobacterial calcification studies so far focused only on few model species and all previous cases had report on extracellular mineralization. In this study, Couradeau et al. identified a new species of cyanobacterium from Lake Alchichica, Mexico. They named this new species Candidatus Gloeomargarita lithophora. They also discover that a new type of biomineralization was performed by this kind of cyanobacteria – intracellular biomineralization. This is perhaps the most exciting discovery in their study and is significantly important for the interpretation of the ancient fossil record.

Dr. Purificación López-García is the team leader of French scientists that works on microbial diversity and evolution. They had collected field samples of the alkaline Lake Alchichica that contained a large diversity of archaea, bacteria and protists. They maintained these field samples long-term in laboratory aquaria. This led them to the observation of phototropic biofilms development on aquarium walls.  They identified this biofilms were dominated by cyanobacteria and one morphotype was particularly abundant. Scanning electron microscopy (SEM) revealed that there are spherical intracellular granules in this morphotype.

The chemical of inclusions were determined by energy-dispersive x-ray spectroscopy (EDXS). The inclusions contained Ca, Mg, Ba and Sr as major elements. The Ba/Ca and Sr/Ca atomic ratios were very high relative to the medium where the cells grow. These ratios implied that this cyanobacterium may have active import systems specific for Sr²⁺ and Ba²⁺ and/or export systems extruding specifically for Ca²⁺.

Then, they used both transmission electron microscopy (TEM) and scanning transmission x-ray microscopy (STXM) to investigate the chemical composition and structure of the Ca-Mg-Sr-Ba inclusions. They found that intracellular inclusions have an unusual stoichiometry that is different from extracellular inclusions.

Next, they used molecular methods to characterize this cyanobacteria strain.  They used cyanobacteria-specific primers to amplify and sequence 16S ribosomal RNA (rRNA) genes. The phylogenetic analysis placed this strain as a member of Gleobacterales, which is a branch that diverged from modern cyanobacteria long time ago.

The cyanobacteria have been suggested that appeared about 2.3 billion years ago on earth since oxygen start to appear in earth atmosphere. However, the oldest fossils of extracellularly-calcified cyanobacteria are dated at only 750–700 million years. There is a huge gap in time between the oldest cyanobacteria and oldest cyanobacteria microfossils founded ad this period called Precambrian Enigma.  If the ancient cyanobacteria formed internal carbonates rather than external precipitation, cells would not be entombed within mineral shells and calcified cyanobacteria may not have formed. Therefore, this would explain why no earlier cyanobacteria microfossils were found.

An Engineered Microbial Platform for Direct Biofuel Production from Brown Macroalgae

This paper was published by researchers in Bio Architecture Lab Company which established in 2007. This company’s main interest is to develop technology to convert seaweed carbohydrates into renewable chemicals and fuels.

Algae has been known as the most cost-effective choice to create biofuel, however more attention was placed on microalgae relatively compared with macroalgae. Macroalgae has passed over as a candidate for biofuel mainly because its primary sugar content is not easily fermented. The breakthrough of this paper is that the authors engineered a microbe that is capable to extract all the major sugars in macroalgae and convert them into fuel and other chemicals.

In this paper, Wargacki et al. presented consolidated bioprocessing of Macroalgae biomass to ethanol by using well-characterized microorganism E.coli. Brown macroalgae is selected as it does not contain lignin and therefore sugars can be released by simple operations such as milling or crushing. The most abundant sugars in brown macroalgae are alginate, mannitol, and glucan. Ethanol fermentation from all three sugar components simultaneously will increased the final ethanol yield. They chose E.coli as a prototype because of its natural ability to metabolize mannitol and glucose.

First, they engineered a secretable Aly enzyme for alginate degradation in E.coli.  Then, they engineered the metabolic pathway for transport and metabolism of alginate oligomers into E. coli. They discovered a 30–kilo–base pair (kbp) DNA fragment that contain all necessary genes for alginate degradation, transport, and metabolism within the genome of Vibrio splendidus 12B01. As direct cloning of a 30-kbp DNA fragment is difficult, they construct fosmid library of random DNA fragments and screened for growth on minimal-salt medium containing alginate oligomers. The designated fosmid was named pALGI. They further cloned few auxillary genes encoded for transport system into pALGI1 to create pALGI3 to improve alginate utilization. At last, they introduced a heterologous homoethanol pathway consisting of Zymomonas mobilis pyruvate decarboxylase (Pdc) and alcohol dehydrogenase B (AdhB) into E.coli to engineer an efficient ethanol-production phenotype.

To test the performance of their microbial platform, they used Saccharina japonica (kombu), a common and widely available brown macroalgae, as a model fermentation substrate. This platform achieved a final titer of about 4.7% volume/volume and a yield of 0.281 weight ethanol/weight dry macroalgae (equivalent to ~80% of the maximum theoretical yield from the sugar composition in macroalgae).

Actin Interacting Protein1 and Actin Depolymerizing Factor Drive Rapid Actin Dynamics in Physcomitrella patens

Tip growth is a mode of polarized cell expansion restricted to a localized region at the tip of the cell (Hepler et al., 2001). Tip growth occurs in algae, fungi and all land plants. The dynamics of the actin cytoskeleton and actin networks (such as F-actin) is essential in controlling tip growth. Numerous actin-binding proteins help to regulate actin’s behaviour.

In this paper, Robert et al. selected Physcomitrella patens as a model system to study the actin binding proteins. They assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF) using reverse genetics, complementation analyses, and cell biological approaches. One of the advantages they chose Physcomitrella patens is because of moss possesses a single ADF gene (Augustine et al., 2008) and a single AIP1 gene.

ADF/cofilins sever actin filaments and result in rapid disassembly and recycling of actin monomer pools (Maciver et al., 1991; Hotulainen et al., 2005; Andrianantoandro and Pollard, 2006; Pavlov et al., 2007). AIP1 enhances ADF/cofilin’s actin filament disassembly activity (Ono, 2003). Previous study has show that AIP1 and ADF/cofilin interact in animals, plants, and fungi (Lida and Yahara, 1999; Okada et al., 1999; Rodal et al., 1999; Allwood et al., 2002).

First, they found a single AIP1 locus using two AIP1 genes sequences from Arabidopsis to BLAST search the P. patens genome and cloned the full-length gene. Then, they assessed the role of AIP1 in plant growth by homologous recombination to remove the moss AIP1 gene. Their result showed that AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. They generated AIP1-mCherry lines and found that AIP1 remains diffusely cytosolic.

To test whether overexpression of ADF can bypass the requirement for AIP1, they transformed ∆aip1 with an ADF expression vector. And they performed a reciprocal experiment to see whether if AIP1 can compensate the loss of ADF by transformed the ADF-RNAi with AIP1 expression vector. It is found that ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. They also found that AIP1 knockout lines accumulate F-actin bundles, which is first reported by Ketelaar et al. (2004) in Arabidopsis.

To examine F-actin behavior in live cells, they imaged cells expressing lifeact-mEGFP (LA) at the cell cortex with spinning disc confocal microscopy or variable angle epifluorescence microscopy (VAEM).  Their data demonstrated that AIP1 is important and ADF is absolutely critical for actin dynamics. Through the imaging data, their also found that the cortical F-actin array have reduced severing frequency in the absence of AIP1, which then decreased the number of available dynamic ends.

In summary, the important finding in this paper is that they demonstrated that ADF is essential for cortical F-actin dynamics and AIP1 enhances ADF’s activity by a mechanism partly dependent on ADF.

One of the question ask from the audience was whether there is homolog of ADF and AIP1 gene in mammalian system. Through web searching, both ADF and AIP1 genes are actually first reported in mammalian cell system.

Structure of HDAC3 bound to co-repressor and inositol tetraphosphate

In 1996, it is a milestone in the history of the epigenetics research field. Charles David Allis cloned the first histone acetyltransferase and published the results in Cell paper. In the same year, Jack Taunton successfully cloned histone deacetylase and published his paper in Science. Histone deacetylase is an enzyme that removes acetyl groups and serves as one of the mechanism for silencing groups of genes. Until now, a number of histone deacetylase enzymes (HDACs) had been identified in eukaryotes. Cancer tumors frequently used HDACs to silence genes that would promote cancer cell death. Therefore, the inhibitor of HDACs is potential anti-cancer drug.

In this paper, the authors report the structure of a complex between human HDAC3 with the deacetylase activation domain (DAD) from the human SMRT co-repressor (also known as NCOR2). HDAC3 belong to class I HDAC and required recruitment into multi-subunit co-repressor complexes for maximal activity. The structure they solved reveals that SMRT-DAD undergoes a major structural rearrangement on forming the complex.

The authors also find that inositol tetraphosphate (Ins(1,4,5,6)P4 or IP4) acting as a  ‘intermolecular glue’ that required for the interaction between SMRT and HDAC3. Their created mutant of key interaction site between Ins(1,4,5,6)P4 and HDAC3 and the mutant lost the deacetylase activity and interaction with SMRT-DAD.

To support their claim that Ins(1,4,5,6)P4 is required for HDAC3 activity, the authors establish an in vitro reconstituition assay. The authors expressed HDAC3 and SMRT-DAD and tested with various concentration Ins(1,4,5,6)P4, Ins(1,4,5)P3, and Ins(1,2,3,4,5,6)P6. Results showed that HDAC activity was sensitive to inositol phosphate concentration and the authors concluded that Ins(1,4,5,6)P4 is the physiological assembly partner. For this result, they didn’t provide the evidence that the endogenous level of Ins(1,4,5,6)P4 in cell which may reflect the true partner of the complex. But, the authors mentioned that whether Ins(1,4,5,6)P4 levels is regulate in the cell is important question and this is not easy to investigate.

The authors suggested that Ins(1,4,5,6)P4 may be regulating the HDAC activity instead of just an essential structural cofactor. Since some other HDACs share sequence homology with HDAC3, they suggested that inositol phosphates possible to have function in these HDACs also. This idea is possible the novelty in this paper since previously there has been report on the inositol phosphate molecule play important role in protein structure. The previous paper “Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing” published in Science, year 2005.

Meiosis-Specific Noncoding RNA Mediates Robust Pairing of Homologous Chromosomes in Meiosis

Meiosis is an essential process for sexual reproduction in plant and animal. One of the important process in meiosis I is homologous chromosome pairing and recombination. In this paper, the authors study the homologous chromosome pairing process in the fission yeast Schizosaccharomyces pombe by monitoring fluorescent tagged chromosome loci.

Previous study showed that initiation of meiosis in fission yeast is regulated by Mei2, an RNA-binding protein. Mei2 formed a nuclear dot at sme2 locus on chromosome. The noncoding RNAs encode by sme2 gene, MeiRNA-S and MeiRNA-L are required for the recruitment of Mei2 dot formation. In this paper, the authors showed the importance of sme2 gene and suggest a working model of this gene. I divide their results into five parts.

First, the authors showed robust pairing at sme2 locus is recombination-independent. Recombination required double strand break formation. Therefore, the authors created a double mutant of sme2 and rec12, which is a required for DSB formation and checked the pairing frequency.

Then the authors used sme2 deletion mutants and sme2 transposition to other loci to show that sme2 is required for the robust pairing.

Third, they showed that two sme2 loci on nonhomologous chromosome is capable of recognizing each other and have 30% pairing.

Forth, the authors did deletion on promoter region (ΔC and ΔD) of sme2 and demonstrated meiRNAs are necessary for the robust pairing. One weakness of the authors is that they didn’t show the transcript level of ΔC heterozygous and sme-2-m heterozygous on their Northern Blot analysis. They suggest that transcripts from both homologous chromosomes are required for the robust pairing.

Their ΔE and ΔF mutants without polyadenylation site produced longer transcripts. For eukaryotic mRNAs, polyadenylation is known for stabilizing and promoting their translation. However, in yeast, polyadenylation is a marking of RNA for degradation for many non-coding RNAs, including tRNA, rRNA, snRNA, and snoRNA. If the authors would like to show that polyadenylation is important for robust pairing, they should produce transcripts without any polyadenylation and not longer transcripts

At last, the authors showed that meiRNA-L is necessary for recruiting Mei2 protein to the locus by using GFP to visualize RNA transcipts in living cell.

The authors demonstrated that the accumulation of meiRNA-L transcript at the sme2 locus acts as a cis-acting pairing factor and suggested that existence of others undiscovered chromosome loci that act together in this working model. If this model is correct, the main future work will be finding these loci.

One term not explained in the presentation is the horsetail movement. Horsetail movement is the oscillatory movement of the nucleus during meiosis prophase I. This oscillatory movement is led by an astral microtubule array emanating from the spindle pole body, which may facilitate synapsis necessary for efficient meiotic recombination.

Literature reading about systematic measurements of “components” in a model organism.

Selected paper: Maier, et al. (2011). Quantification of mRNA and protein and integration with protein turnover in a bacterium. Mol Syst Biol 7, 511.

Summary:

In this paper, the authors integrated experimentally determined abundance data for mRNA, proteins, and individual protein half-lives from Mycoplasma pneumoniae to study its biological function and cellular responses to environmental perturbations.

First, cellular protein abundances and dynamics were determined using mass spectrometry (MS)-based approach. Measured protein abundance changes in response to osmotic stress, mitomycin-induced DNA damage and heat shock revealed a highly dynamic proteome including specific sets of stress response proteins.

Next, the authors quantified the mRNA copy number from tilling array and deep sequencing. Analysis of mRNA-protein abundance ratios revealed that operon-specific and selective post-transcriptional regulatory mechanisms in M. pneumoniae. They also found that the regulation of gene expression is largely decoupled from protein dynamics.

Using modeling estimation of protein homeostasis, they found that the influence of translation efficiency on protein abundance is 40% higher than the influence of protein turnover. By performing the simulations of transcription–translation, they found that low translation efficiencies and long protein half-life buffers gene expression noise.

In the analysis of protein complex abundances and stoichiometries, they found that 81% of all cellular proteome by mass in M. pneumoniae have interaction partners. Their experiments data also found that several ribosomal proteins of M. pneumoniae are found associated with different protein complexes, which suggest that these ribosomal proteins have multi-function.

Finally, they did a quantitative comparison with the the spirochaete bacterium and human pathogenic L. interrogans.They found that protein abundances reflect the respective lifestyles of L. interrogans and M. pneumoniae. L. interrogans utilizes predominantly fatty acid b-oxidation as carbon source, while M. pneumoniae relies mainly on glycolysis for ATP generation. However the relative abundance ratios of glycolytic enzymes are conserved in both bacteria and this suggest that the metabolic adaption involves global abundance regulation of metabolic pathways, rather than the alteration of individual enzymatic activities.

Propose how to analyze/utilize the data/resources:

The authors found a set of 21 proteins with changed abundances in response to all tested perturbations and some proteins were previously unknown for their involvement in general stress response. This indicates that potentially new candidate proteins functioning in stress response can be found and further functional study can be done to validate the results.

Their mRNA-protein abundance analysis result suggest that operon-specific and selective post-transcriptional regulatory mechanisms in M. pneumoniae.  They also found that the regulation of gene expression is largely decoupled from protein dynamics. In additional, protein translocase subunit SecA and partitioning protein ParA lacking a defined heat-shock promoter, also suggesting a possible regulatory mechanism on mRNA stability.  This suggests that the regulatory network of gene, mRNA and protein are far more complexes and study can be done to understand the correlation between them.  This also suggest that the study of cellular responses of organism to stress need to have an integrated view at whole system level.

In their study on mRNA and proteins changes in response to cellular stress, may be it is possible to separate them into two groups: early responses and late responses gene. This will probably enable them to understand more the regulation of gene expression and protein dynamics as organisms may have short-term and long-term strategies to overcome environmental perturbations.

Their experiments data found that several ribosomal proteins have multi-function. Comparative analysis between M. pneumoniae. and L. interrogans also suggest that the metabolic adaption involves global abundance regulation of metabolic pathways. All these results give a new view in evolution study and research on these correlations may be done.

At last, their data could be served as a reference point for future integrative large-scale quantitative studies in other organisms.